ANGPTL7, a therapeutic target for increased intraocular pressure and glaucoma

Glaucoma is a leading cause of blindness. Current glaucoma medications work by lowering intraocular pressure (IOP), a risk factor for glaucoma, but most treatments do not directly target the pathological changes leading to increased IOP, which can manifest as medication resistance as disease progresses. To identify physiological modulators of IOP, we performed genome- and exome-wide association analysis in >129,000 individuals with IOP measurements and extended these findings to an analysis of glaucoma risk. We report the identification and functional characterization of rare coding variants (including loss-of-function variants) in ANGPTL7 associated with reduction in IOP and glaucoma protection. We validated the human genetics findings in mice by establishing that Angptl7 knockout mice have lower (~2 mmHg) basal IOP compared to wild-type, with a trend towards lower IOP also in heterozygotes. Conversely, increasing murine Angptl7 levels via injection into mouse eyes increases the IOP. We also show that acute Angptl7 silencing in adult mice lowers the IOP (~2–4 mmHg), reproducing the observations in knockout mice. Collectively, our data suggest that ANGPTL7 is important for IOP homeostasis and is amenable to therapeutic modulation to help maintain a healthy IOP that can prevent onset or slow the progression of glaucoma.

neuroretinal rim thinning, excavation, notching or nerve fiber layer defects, optic nerves 25 asymmetry or a cup to disc ratio between eyes greater than 0.2. Additional controls for POAAGG 26 were identified from the Penn Medicine Biobank as individuals without ICD-9 diagnoses for 27 glaucoma. 28 29 MALMO: Glaucoma cases were defined as individuals with ≥ 1 in-patient and ≥ 2 outpatient 30 diagnoses for ICD8: 375, ICD9: 365 or ICD10: H40. Individuals with only 1 outpatient diagnosis 31 were excluded from the analysis. Controls were individuals who were not cases or excluded. 32 33 EstBB: Glaucoma cases were defined as individuals with at least 2 records of ICD-10 H40 (and its 34 descendants) and controls were individuals that without a diagnosis for ICD-10 H40-H42, ICD-10 35 H44.5 and ICD-10 Q15.0. Individuals with only 1 ICD-10 H40 code were excluded from the 36 analysis. 37 38 HUNT: Glaucoma cases were defined as individuals with an ICD-10 H40 or an ICD-9 365 39 diagnosis code at 2 separate outpatient encounters or 1 in-patient encounter. Individuals with only 40 1 outpatient encounter were excluded from the analysis. Individuals were excluded from controls 41 if they had any of the following codes: ICD-10 H40-H42, ICD-10 H44.51, ICD-10 Q15.0 / ICD-42 9 365, ICD-9 377.14 or ICD-9 360.42. wards and outpatient clinics (from 1994). Glaucoma cases were defined as individuals with ICD-48 10 H40 and/or ICD8 375, and controls were participants without any of these codes. 49 50 FinnGen: The FinnGen analysis used was finngen_r3_H7_GLAUCOMA. Glaucoma cases were 51 defined as individuals with ICD-10 codes of H40-H42 in the electronic health records and controls 52 were individuals without any of these codes. 53 54

Exome sequencing 55
High coverage whole-exome sequencing was performed at the Regeneron Genetics Center (50, 56 51). NimbleGen probes (VCRome) or a modified version of the xGen design available from 57 Integrated DNA Technologies (IDT) were used for target sequence capture. Sequencing was 58 performed using 75-bp paired-end reads on Illumina v4 HiSeq 2500 or NovaSeq instruments. 59 Sequencing had a coverage depth (ie, number of sequence-reads covering each nucleotide in the 60 target areas of the genome) sufficient to provide greater than 20x coverage over 85% of targeted 61 Lab of the Institute of Genomics, University of Tartu using the Illumina Global Screening Arrays 118 (GSAv1.0, GSAv2.0, and GSAv2.0_EST). At the time of this study altogether 155,772 samples 119 were genotyped and then PLINK format files were created using Illumina GenomeStudio v2.0.4. 120 During the quality control all individuals with call-rate < 95% or mismatching sex that was defined 121 based on the heterozygosity of X chromosome and sex in the phenotype data, were excluded from 122 the analysis. Variants were filtered by call-rate < 95% and HWE p-value < 1 × 10 -4 (autosomal 123 variants only). Variant positions were updated to Genome Reference Consortium Human Build 37 124 and all variants were changed to be from TOP strand using reference information provided by Dr. Weinberg equilibrium test P > 10 -15 and linkage-disequilibrium (LD) pruning (1000 variant 149 windows, 100 variant sliding windows and r 2 < 0.9). The association model used in step 2 of 150 REGENIE included as covariates (i) age, age 2 , sex, age-by-sex and age 2 -by-sex; (ii) 10 ancestry-151 informative principal components (PCs) derived from the analysis of a set of LD-pruned (50 152 variant windows, 5 variant sliding windows and r 2 < 0.5) common variants from the array (imputed 153 for the GHS study) data generated separately for each ancestry; (iii) an indicator for exome 154 sequencing batch (GHS: three batches; UKB: six IDT batches); and (iv) 20 PCs derived from the 155 analysis of exome variants with a MAF < 1% also generated separately for each ancestry. 156 common between the two datasets. We further excluded SNPs with MAF < 10%, genotype 162 missingness > 5% or Hardy-Weinberg Equilibrium test P < 10 -5 . We calculated PCs for the 163 HapMap3 samples and projected each of our samples onto those PCs. To assign a continental 164 ancestry group to each non-HapMap3 sample, we trained a kernel density estimator (KDE) using 165 the HapMap3 PCs and used the KDEs to calculate the likelihood of a given sample belonging to 166 each of the five continental ancestry groups. When the likelihood for a given ancestry group was 167 > 0.3, the sample was assigned to that ancestry group. When two ancestry groups had a likelihood 168 > 0.3, we arbitrarily assigned AFR over EUR, Admixed American (AMR) over EUR, AMR over 169 East Asian (EAS), South Asian (SAS) over EUR, and AMR over AFR. Samples were excluded 170 from analysis if no ancestry likelihoods were > 0.3, or if more than three ancestry likelihoods were 171 > 0.3. Results were subsequently meta-analyzed across studies and ancestries using an inverse 172 variance-weighted fixed-effects meta-analysis.

Phenome-wide association analysis for ANGPTL7 pLOF and missense variants 191
We undertook a phenome-wide analysis of the association of an aggregate of pLOF and missense 192 variants in ANGPTL7 with hundreds of continuous traits or disease outcomes in the GHS and UKB 193 studies. Results were available for 24,082 outcomes across the two cohorts. To control for the 194 number of statistical tests performed, associations were considered statistically significant if the 195 association p-value met a Bonferroni correction for 24,082 tests, that is P < 2 × 10 -6 (corresponding 196 to a p-value threshold of 0.05 divided by 24,082 statistical tests). 197 198 Continuous traits and disease outcomes were defined as described below. In the UKB study, for 199 continuous traits, the values of biomarker, imaging variables or other continuous traits measured 200 during one of the UKB visits or their averages within a given study visit or across study visits were 201 used as outcomes. For binary disease outcomes, case status definition required one or more of the 202 following criteria to apply (a) self-reported disease status or use of medication at digital 203 questionnaire or interview with a trained nurse or (b) EHR of inpatient encounters from the UK 204 National Health Service Hospital Episode Statistics database coded using the ICD-10 coding 205 system. For each binary outcome, controls were individuals without any of the criteria for case 206 definition. In the GHS study, for binary disease outcomes, case status definition required one or 207 more of the following criteria to apply: (1) a problem-list entry of the ICD-10 diagnosis code, (2) 208 an inpatient hospitalization-discharge ICD-10 diagnosis code, or (3)

Derivation of mean corneal refractive power and astigmatism from refractometry traits 226
Corneal refractive power and corneal astigmatism were derived from the autorefractometry and 227 keratometry data available in UKB as previously described (72). Briefly, corneal astigmatism was 228 defined corneal power along strong meridian minus corneal power along weak meridian at 3mm 229 astigmatism is defined as the mean cylindrical power between both eyes (73). 231 232

Bulk RNAseq 233
For the eye atlas bulk RNA sequencing, KAPA Stranded mRNA-Seq Kit by Illumina 234 (https://www.illumina.com/) was used for the library preparation. Pippin HT instrument was used 235 to select 400-600bp fragments prior to sequencing on Illumina Hiseq 2500 using pair-end 2*100 236 base pair protocol. The sequence alignment was performed using ArrayStudio RNA-seq pipeline 237 (https://www.qiagen.com/). More specifically, Human.B37.3 was used as genome reference and 238 OmicsGene20130723 was used as gene model. 239

Generation of Angptl7 -/mice 241
The genetically engineered Angptl7-/mouse strain was created using Regeneron's VelociGene 242 technology (74, 75). Briefly, mouse embryonic stem cells (50% C57BL/6NTac; 50% 243 129S6/SvEvTac; and Crb1 +/+ ) were targeted for ablation of a 571 base pair region of the Angptl7 244 locus, beginning 153 base pairs upstream of the start ATG (mm10 chr4: 148,499,500,442). 245 A self-deleting Hygromycin selection cassette was targeted to the deletion for selection in 246 embryonic stem cells. Heterozygous targeted cells were microinjected into 8-cell embryos from 247 Charles River Laboratories Swiss Webster albino mice, yielding F0 VelociMice that were 100% 248 derived from the targeted cells (75). These mice were subsequently bred to homozygosity and 249 maintained in the Regeneron animal facility during the study period. The resistance cassette was 250 removed during F0 breeding using self-deleting technology.